The HIV-1 pre-integration complex (PIC) that comprises of HIV-1 full-length DNA, viral proteins, and host proteins is essential for viral integration and replication. Previous studies have identified and characterized several human proteins that interact with HIV-1 integrase and play important roles in viral infection. However, the technical limitations in acquiring sufficient amounts of catalytically active PICs has hindered a comprehensive identification of host proteins associated with the HIV-1 PICs. We recently developed a novel approach and identified 18 new human proteins that are specifically associated with the HIV-1 PICs isolated from infected CD4+ T cells. Our preliminary study suggested that one of these proteins, non-POU domain-containing octamer-binding protein (NonO), is required for efficient HIV-1 infection in CD4+ T cells. However, it is unclear whether the other 17 PIC- associated host proteins that we identified can affect HIV-1 infection. NonO is a multifunctional nuclear protein involved in transcription regulation, mRNA splicing, and DNA repair in the cell, but the mechanism by which NonO affects HIV-1 infection is not known. We seek to address these two significant questions in this R21 proposal. We hypothesize that host proteins (such as NonO) specifically associated with catalytically active PICs in HIV-1-infected cells play a role in viral integration and infection. The following two specific aims are designed o test this hypothesis: Aim 1. To determine the role of novel PIC-associated host proteins in HIV-1 infection. In addition to NonO, we may identify other PIC-associated host proteins among these 18 candidates that can significantly affect HIV-1 infection in Aim 1. We will select 2-3 protein candidates that have the most significant effect on viral infection to further study their mechanisms of action. Aim 2. To investigate the mechanisms by which PIC-associated host proteins affect HIV-1 infection. We will first investigate the mechanism by which NonO affects HIV-1 infection, and we will then perform similar studies to examine other protein candidates identified in Aim 1. The long-term goal of this project is to understand the function and mechanism of PIC-associated host proteins in HIV-1 integration or infection and to develop novel therapeutic strategies. An emerging anti-HIV therapeutic strategy is to block the interactions between HIV-1 integrase and its critical cellular cofactors. Our proposed study has the potential to identify novel drug targets to block HIV-1 integration and infection.